BIOCHEMISTRY TRANS 10a Nucleotide metabolism (Part 1).pdf

1A BIOCHEMISTRY NUCLEOTIDE METABOLISM DR. JANDOC I. NUCLEOTIDE METABOLISM OVERVIEW...

1A BIOCHEMISTRY NUCLEOTIDE METABOLISM DR. JANDOC I. NUCLEOTIDE METABOLISM OVERVIEW II. NUCLEOTIDE STRUCTURE  Ribonucleoside and deoxyribonucleoside phosphates (nucleotides)  essential for all cells.  composed of a nitrogenous base; a pentose  Without them, neither ribonucleic acid monosaccharide; and one, two, or three phosphate (RNA) nor deoxyribonucleic acid (DNA) can groups. be produced - proteins cannot be  nitrogen-containing bases belong to two families of synthesized or cells proliferate. compounds: purines and pyrimidines.  Nucleotides  serve as carriers of activated intermediates A. PURINE AND PYRIMIDINE STRUCTURES in the synthesis of some carbohydrates, lipids, and conjugated proteins (for example,  DNA and RNA uridine diphosphate [UDP]-glucose and  contain the same purine bases: cytidine diphosphate [CDP]-choline)  adenine (A) and guanine (G).  are structural components of several  contain the pyrimidine cytosine (C), but essential coenzymes, such as coenzyme A, differ in second pyrimidine base: flavin adenine dinucleotide (FAD[H2]),  DNA contains thymine (T), RNA nicotinamide adenine dinucleotide contains uracil (U). (NAD[H]), and nicotinamide adenine  T and U differ in that only T has a methyl dinucleotide phosphate (NADP[H]). group.  cyclic adenosine monophosphate (cAMP)  Unusual (modified) bases are occasionally and cyclic guanosine monophosphate found in some species of DNA and RNA (for (cGMP) example, in some viral DNA) and in transfer  serve as second messengers in RNA (tRNA). signal transduction pathways.  Base modifications  play an important role as “energy currency”  methylation, glycosylation, in the cell. acetylation, and reduction.  important regulatory compounds for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes.  purine and pyrimidine bases  synthesized de novo or can be obtained through salvage pathways that allow the reuse of the preformed bases resulting from normal cell turnover. Trans FINALS 10a | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 1 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM  First phosphate group  attached by an ester linkage to the 5 -OH of the pentose, forming a nucleoside 5 - phosphate or a 5 -nucleotide.  type of pentose is denoted by the prefix in the names “5 ribonucleotide” and “5 - deoxyribonucleotide.”  If one phosphate group is attached to the 5 -carbon of the pentose, the structure is a nucleoside monophosphate, like adenosine monophosphate [AMP] also called adenylate).  If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate (for example, adenosine diphosphate [ADP] or triphosphate (for example, adenosine triphosphate [ATP]) results.  The second and third phosphates  each connected to the nucleotide by a “high-energy” bond.  [Note: The phosphate groups are responsible for the negative charges associated with nucleotides and cause DNA and RNA to be referred to as “nucleic acids.”] B. NUCLEOSIDES  Nucleoside  addition of a pentose sugar to a base through a glycosidic bond  If the sugar is ribose, a ribonucleoside is produced, and if the sugar is 2- deoxyribose, a deoxyribonucleoside is produced.  The ribonucleosides of A, G, C, and U  named adenosine, guanosine, cytidine, and uridine, respectively.  Deoxyribonucleosides of A, G, C, and T  have the added prefix, “deoxy-” (for example, deoxyadenosine).  Carbon and nitrogen atoms in the rings of the base and the sugar are numbered separately.  Carbons in the pentose are numbered 1 to 5.  When the 5-carbon of a nucleoside (or nucleotide) is referred to, a carbon atom in the pentose, rather than an atom in the III. SYNTHESIS OF PURINE NUCLEOTIDES base, is being specified. C. NUCLEOTIDES  Atoms of the purine ring  contributed by a number of compounds  Nucleotide  amino acids (aspartate, glycine,  addition of one or more phosphate groups and glutamine), CO2, and N10- to a nucleoside. formyltetrahydrofolate. Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 2 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM  Purine ring the PRPP concentration causes a proportional  constructed primarily in the liver by a series change in rate of the reaction. of reactions that add the donated carbons and nitrogens to a preformed ribose 5- C. SYNTHESIS OF INOSINE MONOPHOSPHATE phosphate.  “parent” purine nucleotide A. SYNTHESIS OF 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE  base is hypoxanthine  Four steps in this pathway require ATP as an energy  5-Phosphoribosyl-1-pyrophosphate (PRPP) source  “activated pentose”  Two steps in the pathway require N10-  participates in the synthesis and salvage of formyltetrahydrofolate as a one-carbon donor purines and pyrimidines.  [Note: Hypoxanthine is found in tRNA.]  catalyzed by PRPP synthetase  X-linked enzyme D. SYNTHETIC INHIBITORS OF PURINE SYNTHESIS  activated by inorganic phosphate and inhibited by purine nucleotides (end  Synthetic inhibitors of purine synthesis product inhibition).  sulfonamides  [Note: The sugar moiety of PRPP is ribose, and,  designed to inhibit the growth of rapidly therefore, ribonucleotides are the end products of dividing microorganisms without interfering de novo purine synthesis. When with human cell functions. deoxyribonucleotides are required for DNA  Other purine synthesis inhibitors synthesis, the ribose sugar moiety is reduced.]  structural analogs of folic acid  methotrexate  used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides and, therefore, of DNA and RNA.  Inhibitors of human purine synthesis  extremely toxic to tissues  specially to developing structures such as in a fetus, or to cell types that normally replicate rapidly, including those of bone marrow, skin, gastrointestinal (GI) tract, immune system, or hair follicles. B. SYNTHESIS OF 5-PHOSPHORIBOSYLAMINE  Individuals taking such anticancer drugs can experience adverse effects,  Amide group of glutamine replaces the  anemia, scaly skin, GI tract disturbance, pyrophosphate group attached to carbon 1 of PRPP. immunodeficiencies, and hair loss.  committed step in purine nucleotide biosynthesis.  glu-tamine:phosphoribosylpyrophosphate amidotransferase,  inhibited by the purine 5 -nucleotides AMP and guanosine monophosphate ([GMP] also called guanylate), the end products of the pathway.  Rate of the reaction  controlled by the intracellular concentration of PRPP.  Note: The concentration of PRPP is normally far below the Michaelis constant (Km) for the amidotransferase. Therefore, any small change in Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 3 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM  these kinases do not discriminate between ribose or deoxyribose in the substrate  ATP  source of the transferred phosphate  present in higher concentrations than the other nucleoside triphosphates.  Adenylate kinase  particularly active in the liver and in muscle, where the turnover of energy from ATP is high.  maintain equilibrium among the adenine nucleotides (AMP, ADP, and ATP).  Nucleoside diphosphates and triphosphates  interconverted by nucleoside diphosphate kinase  broad substrate specificity. E. SYNTHESIS OF ADENOSINE AND GUANOSINE MONOPHOSPHATE  conversion of IMP to either AMP or GMP  uses a two-step, energy-requiring pathway  synthesis of AMP requires guanosine triphosphate  (GTP) as an energy source, whereas the synthesis of GMP requires ATP. G. SALVAGE PAHWAYS FOR PURINES  First reaction in each pathway is inhibited by the end product of that pathway.  provides a mechanism for diverting IMP to 1. SALVAGE OF PURINE BASES TO NUCLEOTIDES the synthesis of the purine present in lesser amounts.  Two enzymes are involved:  If both AMP and GMP are present in  adenine phosphoribosyltransferase (APRT) adequate amounts, the de novo pathway of  hypoxanthine-guanine purine synthesis is turned off at the phosphoribosyltransferase (HGPRT). amidotransferase step.  Both use PRPP as source of the ribose 5- phosphate group. F. CONVERSION OF NUCLEOSIDE MONOPHOSPHATE TO  release of pyrophosphate and its NUCLEOSIDE DIPHOSPHATES AND TRIPHOSPHATES subsequent hydrolysis by pyrophosphatase makes these reactions irreversible.  Nucleoside diphosphates  Adenosine  synthesized from the corresponding  only purine nucleoside to be salvaged. nucleoside monophosphates  phosphorylated to AMP by adenosine  by base-specific nucleoside kinase monophosphate kinases Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 4 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM  Other manifestations  motor dysfunction, cognitive deficits, and behavioral disturbances that include self- mutilation (for example, biting of lips and fingers IV. SYNTHESIS OF DEOXYRIBONUCLEOTIDES  Nucleotides required for DNA synthesis  2 -deoxyribonucleotides  produced from ribonucleoside diphosphates  by enzyme ribonucleotide reductase during the S phase of the cell cycle  the same enzyme acts on pyrimidine ribonucleotides 2. LESCH-NYHAN SYNDROME A. RIBONUCLEOTIDE REDUCTASE  rare, X-linked recessive disorder  associated with a virtually complete deficiency of  Ribonucleotide reductase (ribonucleoside HGPRT. diphosphate reductase)  deficiency results in an inability to salvage  composed of two nonidentical dimeric hypoxanthine or guanine, subunits, R1 and R2  excessive amounts of uric acid, the end  specific for the reduction of purine product of purine degradation, are nucleoside diphosphates (ADP and GDP) produced. and pyrimidine nucleoside diphosphates  lack of this salvage pathway causes (CDP and UDP) to their deoxy forms (dADP, increased PRPP levels and decreased IMP dGDP, dCDP, and dUDP). and GMP levels.  The immediate donors of the hydrogen  glutamine:phosphoribosylpyropho atoms needed for the reduction of the 2 - sphate amidotransferase (the hydroxyl group are two sulfhydryl groups on regulated step in purine synthesis) the enzyme itself, which, during the has excess substrate and reaction, form a disulfide bond decreased inhibitors available, and de novo purine synthesis is increased.  decreased purine reutilization and increased purine synthesis  increased degradation of purines  production of large amounts of uric acid, making Lesch-Nyhan a heritable cause of hyperuricemia.  In patients with Lesch- Nyhan syndrome  Hyperuricemia  frequently results in the formation of uric acid stones in the kidneys (urolithiasis)  deposition of urate crystals in the joints (gouty arthritis) and soft tissues. Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 5 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM  responsible for maintaining a balanced supply of deoxyribonucleotides required for DNA synthesis.  regulation of the enzyme is complex  In addition to the catalytic (active) site, there are allosteric sites on the enzyme involved in regulating its activity 1. ACTIVITY SITES  Binding of dATP to allosteric sites  known as the activity sites on the enzyme  inhibits the overall catalytic activity of the enzyme  therefore, prevents the reduction of any of the four nucleoside diphosphates.  Effectively prevents DNA synthesis and explains the toxicity of increased levels of dATP seen in conditions such as adenosine deaminase deficiency 1. REGENERATION OF REDUCED ENZYME  In contrast, ATP bound to these sites activates the enzyme.  In order for ribonucleotide reductase to continue to produce deoxyribonucleotides, the disulfide bond created during the production of the 2 -deoxy 2. SUBSTRATE SPECIFICITY SITES carbon must be reduced.  source of the reducing equivalents  Binding of nucleoside triphosphates to additional  thioredoxin, a peptide coenzyme of allosteric sites ribonucleotide reductase.  known as the substrate specificity sites on  Thioredoxin the enzyme  contains two cysteine residues separated by  regulates substrate specificity two amino acids in the peptide chain.  causing an increase in the conversion of  two sulfhydryl groups of thioredoxin different species of ribonucleotides to  donate their hydrogen atoms to deoxyribonucleotides as they are required ribonucleotide reductase, forming for DNA synthesis a disulfide bond in the process V. DEGRADATION OF PURINE NUCLEOTIDES 2. REGENERATION OF REDUCED THIOREDOXIN  Degradation of dietary nucleic acids  Thioredoxin  occurs in the small intestine, where a family  must be converted back to its reduced form of pancreatic enzymes hydrolyzes the in order to continue to perform its function nucleic acids to nucleotides  necessary reducing equivalents  Inside the intestinal mucosal cells  provided by NADPH + H+  purine nucleotides are sequentially  reaction is catalyzed by degraded by specific enzymes to thioredoxin reductase nucleosides and free bases, with uric acid as the end product of this pathway  Purine nucleotides from de novo synthesis are degraded in the liver primarily. B. REGULATION OF DEOXYRIBONUCLEOTIDE SYNTHESIS  The free bases are sent out from liver and salvaged by peripheral tissues.  Ribonucleotide reductase Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 6 of 7  BIOCHEMISTRY NUCLEOTIDE METABOLISM Hypoxanthine is oxidized by xanthine oxidase to xanthine, A. DEGRADATION OF DIETARY NUCLEIC ACIDS IN THE SMALL which is further oxidized by xanthine oxidase to uric acid, the INTESTINE final product of human purine degradation.  Ribonucleases and deoxyribonucleases  secreted by the pancreas  hydrolyze dietary RNA and DNA to oligonucleotides.  Oligonucleotides  are further hydrolyzed by pancreatic phosphodiesterases  producing a mixture of 3 - and 5 - mononucleotides.  In intestinal mucosal cells  family of nucleotidases removes the phosphate groups hydrolytically, releasing nucleosides that are further degraded by nucleosidases (nucleoside phosphorylases) to free bases plus (deoxy) ribose 1- phosphate.  Dietary purine bases  are not used to any appreciable extent for the synthesis of tissue nucleic acids  Instead, they are generally converted to uric acid in intestinal mucosal cells.  Most of the uric acid enters the blood and is eventually excreted in the urine  Mammals other than primates express urate oxidase (uricase) which cleaves the purine ring, generating allantoin.  Modified recombinant urate oxidase is now used clinically to lower urate levels. A. AMMONIA SOUR B. FORMATION OF URIC ACID An amino group is removed from AMP to produce IMP by AMP deaminase or from adenosine to produce inosine (hypoxanthine-ribose) by adenosine deaminase. IMP and GMP are converted into their nucleoside forms (inosine and guanosine) by the action of 5 -nucleotidase. Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine.  [Note: A mutase interconverts ribose 1- and ribose 5-phosphate.] Guanine is deaminated to form xanthine. Trans FINALS 10b | Abacco, Alderite, Asistin, Balanza, Bayas, Biang 7 of 7